Recently, as demand for high-purity protein medicaments is suddenly increased due to increase of incurable diseases and improvement of public medical standard, relative importance of medicinal recombinant proteins in the health-related bioengineering field is highly raised.
Therefore, the frequency of use of yeast which is a monocellular eukaryotic microorganism as a host system for mass-production of a recombinant protein is gradually increased. Particularly, since yeast has the protein secretion route very similar to those of higher animal cells, it is habitually used as a microorganism host system for production of human-derived secretion proteins. Also, since most kinds of yeast normally secret a very small number of proteins out of the cell, advantageously, recombinant proteins secreted from yeast can be readily recovered and purified. In recent, mass-production of serum proteins, vaccines and other various important medical proteins using non-traditional yeasts including Hansenula polymorpha and Pichia pastoris, other than the traditional yeast Saccharomyces cerevisiae has been successively conducted (Gellissen G., Appl. Microbiol. Biotechnol. 54, 741 (2000)).
As a eukaryotic microorganism, yeast secretes proteins by the substantially same method with mammal cells and involves similar protein modification and cleavage procedures. A protein which has undergone the secretion route becomes to have its final 3-dimensional structure at the Endoplasmic reticulum. In case of glycoprotein, N- and O-bonding sugar chains are attached thereto. Subsequently, the protein is transferred to the Golgi apparatus, in which it is further subjected to the protein modification procedures such as trimming of oligosaccharide or protein cleavage, and thereafter, is transferred to different organs, inserted into components of the cell membrane or secreted out of the cell.
As described above, since the protein secretion procedures in yeast involve various kinds of post-translational modification processes, the secretion and production of a foreign protein in yeast may cause many problems. Particularly, when a recombinant protein is secreted and produced in yeast, it is necessary to use an efficient expression and secretion system in order to increase productivity, but is also important to prevent decomposition of the produced and secreted foreign protein. If a recombinant yeast is cultured for a long period of time at a high concentration in a fermenter, proteases which are naturally secreted from the host cell or exist in the cell through cell lysis are released to medium and degrade the produced recombinant proteins, thereby causing reduction in overall productivity of the recombinant proteins. In order to solve this problem, for yeasts including Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris and the like, which have been used as recombinant protein expression systems, various protease deficient strains have been developed. Primarily, strains, in which PEP4, PRB1, or CPY genes encoding degradative enzymes existing in yeast vacuole are destroyed (Alvarez et al., J. Biotechnol. 38, 81 (1994); Chen et al., Curr. Genet. 27, 201 (1995); Gleeson et al., Methods Mol. Biol. 103, 81 (1998); Kang et al. In Hansenula polymorpha (ed. Gellissen G.) p. 124 (2001)), have been developed. In addition to the vacuole degradative enzymes, kex1Δ strain have been developed, in which KEX1 gene encoding carboxypeptidase α existing in the Golgi apparatus is destroyed. By the kex1Δ strain, C-end decomposition of hirudin in Saccharomyces cerevisiae (Hinnen et al., In Gene expression in recombinant microorganisms (ed. Smith A.), p 121 (1995)), of human epidermal growth factor in Hansenula polymorpha (Heo et al., Protein expr. purif. 24, 117 (2001)) and of rodent or human endostatin in Pichia pastoris (Boehm et al., Yeast 15, 563-567 (1999)) can be significantly reduced.
Recently, yeast aspartic protease type yapsins having activity to recognize and cut basic amino acids existing as a single or a pair in Saccharomyces cerevisiae have been identified, which are novel proteases existing in the cell membrane (Egel-Mitani et al., Yeast 6, 127-137 (1990)). Yapsin1 (also previously known as yeast aspartic protease 3 (YAP3)) was firstly known to the public among the yeast aspartic proteases, and yapsin2 (also previously known as MKC7) was known thereafter (Komano and Fuller, Proc. Natl. Acad. Sci. USA 7, 92, 10752-10756 (1995)). By the Saccharomyces cerevisiae genome information which has been recently disclosed to the public, additional genes encoding at least 5 yapsin type protease presumed to have similar functions, such as yapsin3, yapsin6 and yapsin7, have been reported to exist so far (Olsen et al. Biochem. J. 339, 407-411 (1999)). Though the physiological functions of these yapsins are not clearly shown, as the number of study cases reporting that target recombinant proteins which are intended to secret and produce in S. cerevisiae are cleaved by the protease activity of yapsin is increased, yapsin deficient yeast strains attract public attention as an useful strain for production of a recombinant protein, particularly a foreign peptide having a basic amino acid. Recombinant proteins which have been reported to have problems of being cleaved by yapsins in secretion and production in S. cerevisiae, till now, include human serum albumin (Kerry-williams et al., Yeast 14, 161-169 (1998)), human parathyroid hormone (Kang et al., Appl Microgiol Biotechnol., 50, 187-192 (1998)); Korean Patent Registration No. 0246932 (publicated on Dec. 8, 1999)), insect diuretic hormone (Copley et al., Biochem J., 330, 1333-1340 (1998)), glucagon and glucagon-like peptide (Egel-Mitani et al., Enzyme Microb Technol. 26, 671-677 (2000): U.S. Pat. No. 6,110,703) and human elafin precursor (Bourbonnais et al., Protein Exp. Purif. 20, 485 (2000)). Meanwhile, considering that YPS1 deficient S. cerevisiae strain shows a considerable progress in decomposition of hPTH at the last stage of the cultivation using a fermenter, the present inventors have developed S. cerevisiae yapsin multiple deficient mutant strain (yps1Δ/yps2Δ/yps3Δ), in which the YPS2 and YPS3 genes coding for yapsin2 and yapsin3 are removed. As a result, we have obtained an excellent result of preventing 90% or more of degradation of human parathyroid hormone observed in a high-concentration cultivation (Korean Patent Application No. 2000-51267 and International Application No. PCT/KR01/01447).
H. polymorpha, one of methanol-utilizing yeasts, is in the spotlight as a very useful yeast host for mass production of recombinant proteins since it has advantages in that strong and controllable promoters are developed, alike Pichia pastoris, and a foreign gene can be multiply introduced into the host chromosome (Faber et al., Yeast 11, 1331 (1995)). Up to date, various kinds of foreign proteins have been expressed and the expression levels often reached over 1 g/L in case of high-concentration cultivation using a fermenter. Particularly, it has been reported that when recombinant phytase is secreted and produced, the expression level is about 13.5 g/L (Mayer et al., Biotechnol. Bioeng. 63, 373-381 (1999)). Therefore, the H. polymorpha expression system becomes distinguished as one of the most potential systems among several presently available eukaryotic cell expression systems. Especially, since some of the recombinant proteins which have been produced in the initial stage in H. polymorpha have already passed clinical trials and are on the market (ex., hepatitis B vaccine) or in the product development phase (ex., hirudin), H. polymorpha is considered as a suitable expression system for production of a recombinant protein to be developed as an medicament (Gellissen G., Appl Microbiol Biotechnol. 54 741-750 (2000)). Also, as recently getting into the post-genome era, there is an increased need for a high-efficiency expression system for functional analysis of novel genes, and thus it is expected that an expression system using H. polymorpha would bear a great part in functional and structural analysis of novel proteins as well as mass production of useful proteins derived from higher eukaryotic cells.